Clonality, polyploidy and spatial population structure in Baltic Sea Fucus vesiculosus

Abstract Genetic characteristics of populations can have substantial impacts on the adaptive potential of a species. Species are heterogeneous, often defined by variability at a range of scales including at the genetic, individual and population level. Using microsatellite genotyping, we characterize patterns underlying the genetic heterogeneity in marine macroalga Fucus vesiculosus, with a particular focus on two forms: attached and free‐living. Here we demonstrate that sympatric populations representing the two forms display marked differences in characteristics of reproduction and genetic diversity. Asexual reproduction was ubiquitous in the free‐living form despite being almost entirely absent in the attached form, while signals of polyploidy were common in both forms despite the distinct reproductive modes. Gene flow within and between the forms differed, with barriers to gene flow occurring between forms at various spatial scales due to the reproductive modes employed by individuals of each form. The divergent genetic characteristics of F. vesiculosus demonstrate that intraspecific differences can influence the properties of populations with consequential effects on the whole ecosystem. The differing genetic patterns and habitat requirements of the two forms define separate but closely associated ecological entities that will likely display divergent responses to future changes in environmental conditions.

Alongside the reproductive mode, polyploidization (wholegenome multiplication) is an important variable affecting population divergence and gene flow (Brown & Young, 2000), and the interaction between asexual reproduction and polyploidization may affect genetic diversity and spatial genetic structure. Polyploidization often causes sterility as has been observed in angiosperms (Meichssner et al., 2021) and in some cases algae (Lewis & Neushul, 1995;Zhang & van der Meer, 1988). However, polyploids are often more vigorous compared to diploid conspecifics (Renny-Byfield & Wendel, 2014) although polyploidization has been seen to pose little to no advantage in some algae (Patwary & van der Meer, 1984;van der Meer & Patwary, 1983;Zhang & van der Meer, 1988). Even if sterile, polyploidization can be favorable, at least in the short term, if linked to improved fitness traits because many polyploids are able to reproduce asexually (Comai, 2005) facilitating the rapid colonization and dominance of new areas (Lasker & Coffroth, 1999;Wulff, 1991).
Thus, polyploids may have a competitive short-term advantage, increasing their representation within the population.
Here we investigate the genetic structure of the Baltic Sea population of the marine macroalga Fucus vesiculosus. Fucus vesiculosus is a perennial, dioecious and facultatively asexual species, which occurs at its range margin in the brackish Baltic Sea (Takolander et al., 2017).
In the Baltic Sea, F. vesiculosus can be found in two forms, the most frequently studied being the epilithic form (hereby referred to as attached), but it is also found as a benthopleustophytic form (hereby referred to as free-living; Figure 1) on any substrate within the photic zone (HELCOM, 2013). Traditionally, the free-living form has been assumed to be entirely formed by asexual reproduction due to the absence (Svedelius, 1901) or sterility (Bauch, 1954;Häyrén, 1949) of receptacles, but this is based on anecdotal observations. Asexual reproduction in attached Baltic Sea F. vesiculosus has been observed in both natural populations (Ardehed et al., 2016;Johannesson et al., 2011;Pereyra et al., 2013;Tatarenkov et al., 2005) and in laboratory experiments . Clonality (synonymous with asexual reproduction [De Meeûs et al., 2007]) has also been observed in several natural attached Fucus populations along the Finnish and Swedish coasts of the Bothnian Sea (Pereyra et al., 2009;Rinne et al., 2018), although the species identity of clonal samples (F. vesiculosus or F. radicans) has not always been clear.
Although clonality within the attached Baltic Sea F. vesiculosus form is more commonly documented, unattached forms are also observed to display clonal growth. An embedded form of F. vesiculosus in the western Baltic Sea near Glücksburg (Germany) reproduces entirely by clonal growth (Meichssner et al., 2021). However, the mode of reproduction is poorly understood in free-living F. vesiculosus.
Nevertheless, the assumptions on the association between asexual reproduction and the free-living form may have some validity.
Clonal growth complicates the definition of an individual, and thus here we briefly define the terminology. Genet refers to the entire clone or genotype, while ramet refers to the unit or module of clonal growth denoting the smallest physiologically integrated F I G U R E 1 An image of an aegagropiloid free-living Fucus vesiculosus thalli from Tvärminne, Finland (a) and a free-living F. vesiculosus population at Askö, Sweden (b).
To our knowledge, however, polyploidy has not been documented in the Baltic Sea F. vesiculosus and all previous genetic studies have assumed a diploid genetic system (e.g. Ardehed et al., 2016;Johannesson et al., 2011;Rinne et al., 2018).
In this work, we investigate the reproductive mode and spatial genetic structure in the Baltic Sea F. vesiculosus population by using DNA microsatellite markers. We focused on the two forms -attached and free living -sampling both forms from the same localities. We compared the amount of genetic variation in the two forms, determined the representation of reproductive modes (sexual vs. asexual reproduction in the form of clonal growth) in the study populations and assessed the spatial genetic structure across the study area. We hypothesized that the attached form reproduces primarily sexually and the free-living form primarily asexually. We also hypothesized spatial structuring among all populations, although connectivity between sympatric populations was predicted to be relativity high. Finally, based on previous studies, we expected that polyploidy would be absent in the Baltic Sea F. vesiculosus, yet we also found a substantial proportion of potential polyploid samples in our study populations. This allowed us to consider the association between genome size and form.

| Data analysis
Alleles were scored using Genemapper 5 (Applied Biosystems™) and checked by eye. A sizable proportion of individuals displayed more than two alleles in at least one of the loci studied. Thorough validity checks were performed including repeat DNA extractions, PCR reactions, and ABI plate setups with new reagents to determine the validity of the third and/or fourth allele. The trend of ≥3 observed alleles in the electropherograms was consistent and repeatable for the apparent polyploid specimens. Allele peaks for apparent polyploid specimens were often of near to equal amplitudes and thus, as accurate allele determination is essential for population genetic analysis, all called alleles were kept for determining the genotype. This is problematic, as the allele dosage of polyploid samples cannot be assessed from the genotype of the sample. For instance, an individual sample appearing as genotype AB may indeed be AB if it is diploid, but AAB or ABB if it is triploid, and AAAB, AABB, or ABBB if tetraploid. As a result, we could not use standard inference of diploid genotypic data, but used Meirmans (2020) software Genodive version 3.05 to infer the ploidy level and dosage compensation of individual samples instead. Ploidy level was discerned by the maximum observed allele count per each sample by the software Genodive version 3.05 (Meirmans, 2020). Missing data were corrected by imputation and a maximum likelihood method was used to correct for the unknown dosage of the alleles using Genodive version 3.05 (Meirmans, 2020) implementing a modified version of the method of De Silva et al. (2005). As accurate estimation of allele frequencies of individuals and populations is the basis for most population genetic analyses, data based on dosage compensation were used here.
Unless otherwise indicated, all analyses were conducted by using Genodive version 3.05 (Meirmans, 2020) and the significance of the estimated parameters was defined with permutation tests.
Clonal lineages were identified using a Stepwise Mutation Model with a threshold of 0 and set clones specific to every population.
Tests for clonal population structure based on the concept of clonal diversity (Gómez & Carvalho, 2000) using Corrected Nei's diversity index were performed during clonal assignment. A total of 1228 multilocus genotypes (MLGs) were identified from the 1443 samples.
Proportions of clones and shared clonal lineages were calculated manually. Clonal diversity was described as expected heterozygosity within populations (H s ; Nei, 1987), the Shannon index (shc; both corrected for sample size [Chao & Shen, 2003]), the effective number of genotypes (eff) and the evenness of genotypes over the population (eve). Moreover, hidden clonal diversity was estimated with a rarefaction analysis using iNEXT Online (Chao et al., 2016)  Two k independent-samples Kruskal-Wallis tests were used to test the significance of ploidy levels across populations and loci. Pearson correlation was used to test the relationship between the number of alleles at a locus and the ability to detect polyploids. Two Mann-Whitney tests were used to test the differences in amounts of total MLGs and clonal MLGs between forms and a one-sample t test was used to determine the difference in the number of clonal MLGs among clonal populations. Chi-square crosstabulation was used to test the association between ploidy level (groups: diploid, polyploid) and clonality.
The following analyses were performed on two datasets, either including a single ramet per clonal lineage per population, or including all ramets, or on both datasets concurrently. Genetic diversity (H s ) was estimated within populations and for the total population. Spatial genetic structure was assessed using several methods. First, spatial structure was described visually by conducting a principal component analysis (PCA), calculated from a covariance matrix (1000 permutations). Second, pairwise genetic differentiation between populations was estimated using Rho ST index, which is analogous to F ST , but independent of the ploidy level (Meirmans, 2020;Ronfort et al., 1998). Significant differentiation (Rho > 0; 1000 permutations) was determined and manually corrected using Bonferroni correction. Third, isolation by distance (IBD) was determined by plotting pairwise genetic differentiation against pairwise geographic distances generated by the Geographic Distance Matrix Generator (Ersts, 2012). Significance of the matrix correlation was tested with Mantel's test (1000 permutations). IBD was assessed for the total data, and for both forms separately. Finally, we described the distribution of genetic variation with hierarchical Analysis of Molecular Variance (AMOVA; Excoffier et al., 1992). In the AMOVA, genetic variation was first allocated to different hierarchical levels and then the associated fixation indices of Rho and their significance (Rho > 0; 999 permutations) were determined. We used two alternative a priori hierarchies in AMOVA: (i) populations were nested within the form; (ii) populations were nested within subbasins. The latter analysis was performed separately for (A) attached and (B) free-living populations.

| Polyploidy
We found potential signals of polyploidy in all populations except one (TZ2.F; Figure 2). The number of samples with ≥3 alleles differed significantly across populations (Kruskal-Wallis H 282.8, df 33, p < .001). The average frequency of potential polyploid samples was 38% (range among sites: 7%-84%) and over half of the samples appeared polyploid in five attached and six free-living populations.
Polymorphism of the loci affected the ability to detect potential polyploids (Appendix S4). There was a significant difference in the ability of each locus to detect ≥3 alleles (Kruskal-Wallis H 1179.928, df 7, p < .001, Appendix S4A) with greater allele variance at a given locus being weakly but non-significantly associated with capturing ≥3 alleles (Pearson correlation .522,N 8,p .184; Appendix S4B). The determined ploidy level of the samples was significantly associated with the type of MLGs observed (Appendix S5). Diploid thalli were more likely to be associated with clonal MLGs than would be expected, while potential polyploids are more likely to be unique MLGs.

| Clonality
Clonal MLGs were found in three attached populations in Askö (AS3

| Intraspecific variation
Genetic diversity across the subbasins (H s ) did not differ significantly (range: 0.58-0.64), but H s differed significantly between the two forms (Table 3). Genetic diversity was greater in the attached population compared to the free-living one, and the difference between the forms was boosted when all ramets of the clonal MLGs were included in the analysis. In the PCA, the first two principal components explained 33% In AMOVA, there was significant spatial structuring at all hierarchical levels, both between forms and among each form (Tables 4   and 5). However, the forms were strikingly different in their pattern of spatial structuring. Differences between populations within the forms explained much more of the total variance than the differences between the forms themselves. Likewise, genetic differentiation (Rho ST ) was an order of magnitude larger within compared to between forms (Table 4)  within subbasins compared to the attached populations. While corroborating that the forms belong to the same species, their varying genetic characteristics suggest that their reproductive modes, dispersal capabilities, and population connectivity differ. Thus, our results highlight the necessity to manage the genetic diversity of each form with independent but congruous efforts.

F. vesiculosus
When using predominantly the same DNA microsatellite markers, our results showed that the genetic diversity was at the same level (H s 0.44-0.69) when compared to previous studies in the Baltic Sea (Johannesson et al., 2011;Pereyra et al., 2013;Tatarenkov et al., 2007) and globally (Perrin et al., 2007;Wallace et al., 2004). As expected based on the assumed dominance of asexual reproduction in the free-living form (Bauch, 1954;Svedelius, 1901), the free-living populations were significantly less genetically diverse than their attached counterparts. Nevertheless, genetic diversity was still within expected limits for the species in both forms. Thus, frequent asexual reproduction in the free-living form did not drastically reduce the overall genetic variation in F. vesiculosus.

| Polyploidy in the Baltic Sea F. vesiculosus
Polyploidy has not been found in previous genetic studies of Baltic Sea F. vesiculosus (Ardehed et al., 2016;Johannesson et al., 2011;Pereyra et al., 2009;Rinne et al., 2018;Tatarenkov et al., 2005Tatarenkov et al., , 2007, but has infrequently been reported elsewhere in Fucus spp.  (Coyer, Hoarau, Pearson, et al., 2006;Gómez Garreta et al., 2010;Sjøtun et al., 2017). Based on these findings, we did not expect to find variation in the ploidy level in our study, yet a considerable portion of our samples were determined to possess ≥3 alleles in at least one locus. We verified this finding by meticulously scrutinizing the multiple-peak patterns in the electropherograms and by reanalysing samples, showing that these patterns were repeatable and consistent.

F I G U R E 4 Proportions of shared clonal lineages in the
In natural populations, tetraploidy is generally the most common polyploid level (Comai, 2005), but instead, we observed only a few tetraploids (4%) and extensive triploidy (35%). It must be noted, however, that our assessment of the ploidy level is based on using genetic markers and higher ploidy levels can be missed tetraploids may not be accurately estimated. As our a priori assumption of F. vesiculosus being diploid in the Baltic Sea appeared incorrect, we could only employ a posteriori methods designed to accommodate polyploid data in mixed-ploidy populations, i.e.
use dosage compensation (Meirmans, 2020). This emphasizes the need to assess the level of ploidy in the samples when polyploidy is suspected, using e.g. micro spectrofluorometry or flow cytometry. Further study is warranted to affirm these signatures of polyploidy within Baltic Sea F. vesiculosus.
Polyploidy can arise as hybridization between two (or more) related species (allopolyploidy), or it can be a result of multiplication of the whole genome of a single parent species (autopolyploidy). In the Baltic Sea, F. vesiculosus, F. radicans and F. serratus are native, and F. evanescens is an invasive species Malm et al., 2001;Wikström et al., 2002), which suggests that both allopolyploid and autopolyploid forms can potentially arise.
Outside the Baltic Sea, Fucus species are typically separated by intertidal zonation (Colman, 1933;Fritsch, 1945;Lubchenco, 1980;Zaneveld, 1937) and species occur at the same geographical sites, whereas in the Baltic Sea, the distribution of different Fucus species is increasingly controlled by the salinity gradient (Isaeus, 2004 In the Gulf of Riga, F. vesiculosus and F. radicans occur both sympatrically and allopatrically (Johannesson et al., 2011;Pereyra et al., 2013), and both allopolyploidy and autopolyploidy are possible origins of polyploidy in this area. There are several pieces of evidence that favor autopolyploidy in the Gulf of Riga populations.
First, other Fucus spp. populations are geographically sufficiently separated from our Gulf of Riga sites; second, as above, genetic differentiation between the Gulf of Riga populations and our other study sites was relatively low; third, the occurrence of polyploidy was analogous to the rest of the populations. Thus, autopolyploidy is a more likely origin of polyploidy also in the Gulf of Riga populations, but further research is needed to confirm this.
Frequent fusion of reduced and unreduced gametes (Bretagnolle and Thompson, 1995) alongside the advantages of polyploidy (heterosis, gene redundancy, asexual reproduction; Comai, 2005) may be supporting the apparent triploidy. Several natural plant populations are known to be dominated by triploids Lee et al., 2016;Mock et al., 2012) and thus the high amounts of observed triploids within our populations are not wholly unexpected. As triploids are unstable and frequently sterile (Ramsey and Schemske, 1998), the many unique MLGs in the attached populations would suggest that the benefits of polyploidy are not large enough to allow for triploids to dominate attached populations. Conflicting this, the high frequency of triploidy in free-living populations appears more logical, because infertile triploids can propagate vegetatively (Pearson, 2001). After a single event of triploid zygote formation, several events of asexual reproduction by the mature triploid thallus may lead to multiple triploid clones within the free-living population. As triploids are often more vigorous with higher fitness compared to diploids (Miller et al., 2012), triploid clones could spread rapidly through the population. However, if polyploids receive a fitness boost, it could be argued that tetraploids should be more abundant as they are both stable and fertile (Comai, 2005), this should apply to both sexually and asexually reproducing populations. Although within algae, increasing the genome size may not to provide a fitness advantage. Polyploid Gracilaria tikvahiae have lower fitness than their diploid conspecifics and increasing genome size (triploidy vs. tetraploidy) results in an even greater reduction of fitness (Patwary and van der Meer, 1984;van der Meer and Patwary, 1983;Zhang and van der Meer, 1988). Thus, the rarity of tetraploidy compared to triploidy may be valid or an artifact resulting from the resolution of the genetic markers used.

| The occurrence of clonality in F. vesiculosus
Many of our study populations included samples that shared their multilocus genotype. As the probability of clonal MLGs arising under random mating within most populations was minimal (Appendix S6), samples sharing genotypes can be considered true clones (ramets).
Thus we describe them as clonal MLGs. As algae are known to asexually reproduce through various methods (e.g. stoloniferous growth, fragmentation, adventitious branches [Collado-Vides, 2001;Fritsch, 1935Fritsch, , 1945) the exact mechanisms underlining clonal production cannot be determined from microsatellite analysis alone. We suggest that the fragility of F. vesiculosus thalli, particularly of the free-living form (R. Preston, pers. comm.), alongside the frequent occurrence of adventitious branches (Kinnby et al., 2019) indicates that fragmentation and/ or adventitious branches are credible methods. As the free-living form has previously been presumed to be sterile (Bauch, 1954;Häyrén, 1949;Svedelius, 1901), the origin of clonal MLGs would therefore have to be either entirely or predominantly through clonal growth.
The between populations and will be discussed below.
Asexual reproduction has been suggested to increase towards the range margins of the species (Billingham et al., 2003;Eckert, 2001;Kearney, 2003). In the studies above, many of the study populations were located north of our study area, in the Bothnian Sea, and the lower salinity of these study sites may have contributed to the higher prevalence of clonal MLGs. However, one of our attached populations with a higher amount of clonal MLGs (AS3) was not among the sites with lowest salinity. Additionally in attached populations at Öland and Öregrund, with similar salinity ranges to our study sites, clonal MLGs were pervasive (Ardehed et al., 2016;Johannesson et al., 2011;Tatarenkov et al., 2005).
Interestingly, both Ardehed et al. (2016) and our study found that clonality was absent for the attached form in the Archipelago Sea. Thus, it is possible that the prevalence of clonality varies due to selective and/or neutral processes (Rafajlović et al., 2017) in attached populations in the Baltic Sea and is rare at our study sites.

| Spatial genetic structure
The Baltic Sea F. vesiculosus population was structured at various levels, with both spatial factors and form influencing the connectivity among populations. When the data were organized according to the forms (populations nested within forms), forms were significantly genetically differentiated from each other. However, this hierarchical level explained only a minor part (1%) of the total variance in the data and the among-populations level explained more than an order of magnitude more (17% , Table 4). When attached and freeliving populations were analyzed separately in the AMOVA, populations were significantly differentiated both within and among the subbasins in both forms, but the spatial structuring among local populations within the subbasins was clearly stronger in the free-living compared to the attached form. Moreover, the attached populations showed a weak but significant isolation-by-distance effect, as expected when gene flow does not cover the whole study area.
Previous results on spatial genetic structuring of the Baltic Sea F. vesiculosus population have been somewhat mixed. Our work is in line with previous results showing strong structure among spatially closely located populations along the Swedish east coast (Pereyra et al., 2009;Tatarenkov et al., 2007), the Finnish west coast (Rinne et al., 2018), and also at larger scales (Ardehed et al., 2016;Johannesson et al., 2011). Contrary to this, a wide-scale study only found significant genetic structuring among local attached F. vesiculosus populations in the Gulfs of Bothnia and Riga, but not among the regions (Pereyra et al., 2013). Previous results on the effects of geographic components on the spatial genetic structure have also been mixed. Tatarenkov et al. (2007) showed an isolation-bydistance signal at both local (<10 km) and large (<1000 km) scales in F. vesiculosus, while (Ardehed et al., 2016) did not find such signal in F. vesiculosus nor F. radicans, although the scale and distribution of these studies were not directly comparable.
Our results on the spatial genetic structuring suggest that the gene flow between the two F. vesiculosus forms is not completely free with the two forms representing different spatial genetic structures. The spatial genetic structure within the free-living form appears to be increasingly driven by the strong genetic differentiation among local free-living populations, while gene flow is equally restricted at both large and small spatial scales in the attached form.
Free-living populations thus appear far more isolated at a local scale indicating geographically close populations may have markedly different origins with genetic differences maintained by clonal growth.
Our results also suggest a difference in gene flow between the two forms. In the attached form, gene flow extends further than in the free-living form and it seems that it is not restricted to the scale of subbasins, which shows as a significant IBD signal. On the contrary, gene flow in the free-living form appears to be random and more The origin of free-living populations is discussed next in more detail.

| Origin and maintenance of free-living F. vesiculosus
It is conceivable that free-living F. vesiculosus populations emerge either by asexual (clonal growth) or sexual reproduction and subsequent dispersal of propagules to a new location. Both modes of reproduction could take place in either attached or free-living populations and moreover, these alternatives are not mutually exclusive. Our study does not provide direct answers to the origin of the free-living populations, but clonal diversity and distribution, and the spatial structure of the population allow us to discuss these scenarios. Freeliving forms of Fucus spp. populations have classically been assumed to derive from attached populations through clonal growth whereby pieces of thalli (either as pieces of typical adult thalli or adventitious branches) would detach from attached individuals, float freely and eventually aggregate in still locations and persist over a longer time (Bauch, 1954;Cotton, 1912;Den Hartog, 1959;Fritsch, 1945;Häyrén, 1949;Luther, 1981;Svedelius, 1901). Alternatively, pieces of thalli may detach from other free-living populations and eventually aggregate in a new location.
Another scenario for the origin of free-living populations involves sexual reproduction in attached populations and, theoretically, also in free-living populations. Instead of attaching to the substratum, zygotes could attach to an inadequate anchoring surface leading to subsequent detachment (Baker and Bohling, 1916;Boney, 1966;Chapman and Chapman, 1973;Fritsch, 1945) or they may settle in a quiet environment where attachment substrates are unavailable and develop in situ (Lee, 1989). The typically soft sediment dominated coastal environments associated with free-living populations provide unfavorable conditions for colonization from small sexually recruited stages due to the effects of sedimentation including burial, altered light conditions and altered chemical micro-environment (Berger et al., 2003;Chapman and Fletcher, 2002;D'Antonio, 1986;Daly and Mathieson, 1977;Devinny and Volse, 1978;Eriksson and Johansson, 2003). In fact, recruitment of new F. vesiculosus in soft sediment dominated environments has previously been unsuccessful (Shaughnessy, 1982 would be expected to be locally differentiated (Neiva et al., 2012) and dominated by one to a few clones as a signature of recurrent asexual reproduction (Barrett, 2015).  (Rothäusler et al., 2015(Rothäusler et al., , 2020, but this scenario could be verified only by extending the study area considerably. It seems that once a free-living population has emerged, it is maintained in multiple ways. Clonal growth is recurrent within the free-living populations, but additional supply from other populations must also be relatively frequent. Overall it seems that free-living populations are heterogeneous and processes maintaining the populations vary. To conclude, attached populations predominantly reproduce sexually, and connectivity among populations is often higher than in the free-living form. This suggests that the attached populations are able to maintain genetic diversity and adaptive potential in the face of changing environmental conditions as a network of populations. Conversely, free-living populations are often dominated by clonal lineages with many populations showing greater isolation, resulting in decreased genetic diversity. Nevertheless, clonally dominated free-living populations may benefit temporarily from the predicted changes in the salinity regimes and increasing temperature within the Baltic Sea (Meier et al., 2021). If one genotype is particularly well-suited to the new conditions, asexual reproduction would allow rapid colonization by this genotype (Lasker and Coffroth, 1999;Wulff, 1991). In the long run, it is more likely that populations deprived of genetic diversity are vulnerable, as clonally reproducing organisms often are less able to adapt to changing environments (Nieuwenhuis and James, 2016). Free-living populations are more unstable than their attached counterparts because the free-living form is generally found in sheltered, shallow areas, often close to shore, which are particularly vulnerable to the effects of environmental change and eutrophication (Brito et al., 2012). Whole populations have also been lost in unfavorable flood situations (Bauch, 1954) or winter storms (Norberg, 1995), which have also been predicted to increase under climate change (Meehl et al., 2007). Consequently, free-living populations show an increased vulnerability to local extinctions compared to the attached populations, emphasizing the need to consider the form independently.

ACK N OWLED G M ENTS
We are grateful to Sven Dahlke and Irmgard Blindow (University of Greifswald, Germany) for obtaining samples from the Biological Station Hiddensee. We also wish to thank the staff, particularly Kirsi Kähkönen, at the MES laboratory, University of Helsinki, for their advice and assistance with the microsatellite genotyping.

FU N D I N G I N FO R M ATI O N
Funding for this project was provided through grants from the Walter and Andrée de Nottbeck Foundation and the Onni Talas Foundation.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.